Silencing of ZFP36L2 increases sensitivity to temozolomide through G2/M cell cycle arrest and BAX mediated apoptosis in GBM cells

Abstract

Despite the advancements in primary brain tumour diagnoses and treatments, the mortality rate remains high, particularly in glioblastoma (GBM). Chemoresistance, predominantly in recurrent cases, results in decreased mean survival of patients with GBM. We aimed to determine the chemosensitisation and oncogenic characteristics of zinc finger protein 36–like 2 (ZFP36L2) in LN18 GBM cells via RNA interference (RNAi) delivery. We conducted a meta-analysis of microarray datasets and RNAi screening using pooled small interference RNA (siRNA) to identify the druggable genes responsive to GBM chemosensitivity. Temozolomide-resistant LN18 cells were used to evaluate the effects of gene silencing on chemosensitisation to the sub-lethal dose (1/10 of the median inhibitory concentration [IC50]) of temozolomide. ZFP36L2 protein expression was detected by western blotting. Cell viability, proliferation, cell cycle and apoptosis assays were carried out using commercial kits. A human apoptosis array kit was used to determine the apoptosis pathway underlying chemosensitisation by siRNA against ZFP36L2 (siZFP36L2). Statistical analyses were performed using one-way analysis of variance; p > 0.05 was considered significant. The meta-analysis and RNAi screening identified ZFP36L2 as a potential marker of GBM. ZFP36L2 knockdown significantly induced apoptosis (p < 0.05). Moreover, ZFP36L2 inhibition led to increased cell cycle arrest and decreased cell proliferation. Downstream analysis showed that the sub-lethal dose of temozolomide and siZFP26L2 caused major upregulation of BCL2-associated X, apoptosis regulator (BAX). ZFP36L2 has oncogenic and chemosensitive characteristics and may play an important role in gliomagenesis through cell proliferation, cell cycle arrest and apoptosis. This suggests that RNAi combined with chemotherapy treatment such as temozolomide may be a potential GBM therapeutic intervention in the future.

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Funding

The study was funded by the Higher Institution Centre of Excellence (HICoE) (Grant No. JJ-008-2011) from the Ministry of Higher Education, Malaysia.

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MFCM: Performed all experiments and wrote the first draft of the manuscript; EAMH: contributed equally to the writing of the manuscript; NAAM and RH: supervised MFCM and provided critical comments on the manuscript; KI: data analysis; RJ: provided the HICoE grant and critical comments on the manuscript.

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Correspondence to Roslan Harun or Rahman Jamal.

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Che Mat, M.F., Mohamad Hanif, E.A., Abdul Murad, N.A. et al. Silencing of ZFP36L2 increases sensitivity to temozolomide through G2/M cell cycle arrest and BAX mediated apoptosis in GBM cells. Mol Biol Rep (2021). https://doi.org/10.1007/s11033-021-06144-z

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Keywords

  • Glioblastoma
  • RNAi screening
  • Temozolomide
  • Chemosensitisation
  • ZFP36L2